Microparticles with adsorbed polypeptide-containing molecules

ABSTRACT

Microparticles with absorbed polypeptide-containing molecules formed without the use of surfactant, methods of making such microparticle compositions, and uses thereof, are disclosed. The microparticles comprise a polymer, such as a poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and the like. Preferred polymers are poly(D,L-lactide-co-glycolides), more preferable those having a lactide/glycolide molar ratio ranging from 40:60 to 60:40 and having a molecular weight ranging from 20,000 Daltons to 70,000 Daltons. Preferred polypeptide containing molecules are bacterial and viral antigens (including HIV antigens, meningitis B antigens, streptococcus B antigens, and Influenza A hemagglutinin antigens).

STATEMENT OF RELATED APPLICATION

This application is a 371 of PCT/US03/05017, filed Feb. 20, 2003, claimsthe benefit of U.S. Provisional Patent Application Ser. No. 60/358,315,filed Feb. 20, 2002, entitled “Microparticles With AdsorbedPolypeptide-Containing Molecules,” each of which is hereby incorporatedby reference.

TECHNICAL FIELD

The present invention relates generally to pharmaceutical compositions.In particular, the invention relates to biodegradable microparticleswith adsorbed polypeptide-containing molecules that are formed withoutthe use of surfactant, methods for preparing such microparticles, anduses thereof.

BACKGROUND

Particulate carriers have been used with adsorbed or entrapped antigensin attempts to elicit adequate immune responses. Such carriers presentmultiple copies of a selected antigen to the immune system and promotetrapping and retention of antigens in local lymph nodes. The particlescan be phagocytosed by macrophages and can enhance antigen presentationthrough cytokine release.

For example, commonly owned International patent application WO 98/33487(PCT/US98/01738) and co-pending U.S. patent application Ser. No.09/015,652, filed Jan. 29, 1998, describe the use of antigen-adsorbedand antigen-encapsulated microparticles to stimulate immunologicalresponses, including cell-mediated immunological responses, as well asmethods of making the microparticles. Polymers used to form themicroparticles include poly(lactide) and poly(lactide-co-glycolide),also referred to herein as “PLG”.

Commonly owned International patent application WO 00/06123(PCT/US99/17308) and co-pending U.S. patent application Ser. No.09/715,902 disclose methods of making microparticles having adsorbedmacromolecules, including polynucleotides and polypeptide antigens. Themicroparticles comprise, for example, a polymer such as apoly(alpha-hydroxy acid) (e.g., PLG, a polyhydroxy butyric acid, apolycaprolactone, a polyorthoester, a polyanhydride, and the like) andare formed using, for example, cationic, anionic or nonionic detergents.Microparticles containing anionic detergents, such as PLG microparticleswith sodium dodecyl sulfate (SDS), are proposed for the use ofpositively charged macromolecules, such as polypeptides. Microparticlescontaining cationic detergents, such as PLG microparticles with CTAB(also known as cetrimide or cetyl trimethyl ammonium bromide), areproposed for the use of negatively charged macromolecules, such as DNA.The use of such microparticles to stimulate immunological responses,including cell-mediated immunological responses, is also disclosed.

In each of the above references, however, one or more surfactants areutilized during preparation of the macromolecule-adsorbedmicroparticles. Unfortunately, the use of surfactants can raisetoxicological issues that result in additional regulatory scrutinyduring product registration, among other consequences.

SUMMARY OF THE INVENTION

The present inventors have unexpectedly found that microparticles withadsorbed polypeptide-containing molecules can be formed in the absenceof a surfactant.

For instance, according to a first aspect of the invention, abiologically active microparticle composition is provided, whichcomprises: (a) microparticles comprising a polymer selected from thegroup consisting of a poly(α-hydroxy acid), a polyhydroxy butyric acid,a polycaprolactone, a polyorthoester, a polyanhydride, and apolycyanoacrylate; and (b) a polypeptide-containing molecule, which isadsorbed to the microparticles. The composition is formed in the absenceof anionic surfactant, and is preferably formed in the absence of allsurfactants, including anionic, cationic, nonionic and zwitterionicsurfactants.

Preferred polymers are poly(α-hydroxy acids), more preferably thoseselected from the group consisting of poly(L-lactide), poly(D,L-lactide)and poly(D,L-lactide-co-glycolide). More preferred arepoly(D,L-lactide-co-glycolide) polymers. Preferredpoly(D,L-lactide-co-glycolide) polymers are those having alactide/glycolide molar ratio ranging from 25:75 to 75:25, morepreferably 40:60 to 60:40, and having a molecular weight ranging from10,000 to 100,000 Daltons, more preferably from 30,000 Daltons to 70,000Daltons.

Preferred biologically active polypeptide-containing molecules includebacterial and viral antigens. HIV antigens (such as g41, gp120, gp140,p24gag and p55gag antigens), meningitis B antigens (such as meningitis Brecombinant protein 287 antigen), streptococcus antigens (such as groupB streptococcus antigen), and Influenza A hemagglutinin antigens areparticularly preferred.

In some embodiments, the microparticle composition is provided with afurther biologically active macromolecule, which may be bound or unboundto the microparticles, and may even be entrapped within the polymer. Forexample, the microparticle composition may be provided with an adjuvant,particularly a Th1 stimulating adjuvant. Preferred adjuvants include CpGoligonucleotides, LTK63, LTR72, MPL, aminoalkyl glucosaminide4-phosphates (AGP's), imidazoquinoline adjuvants, lipopolysaccharidemimetic adjuvants, QS21, double-stranded RNA (dsRNA) and aluminum salts,including aluminum phosphate.

According to another aspect of the present invention, a pharmaceuticallyacceptable excipient is added to the above microparticle compositions.

Another aspect of the invention is directed to the delivery of apolypeptide-containing molecule to a vertebrate subject, which comprisesadministering to a vertebrate subject the above microparticlecomposition.

In other aspects of the invention, the above microparticle compositionsare used in the diagnosis of diseases, in the treatment of diseases, invaccines, and/or in raising an immune response.

For example, in an additional aspect, the invention is directed to amethod for eliciting a cellular and/or humoral immune response in avertebrate subject, which comprises administering to a vertebratesubject a therapeutically effective amount of a microparticlecomposition as described above.

Another aspect of the invention is directed to a method of immunization,which comprises administering to a vertebrate subject a therapeuticallyeffective amount of the microparticle composition above.

Still other aspects of the invention are directed to methods ofproducing microparticles. In general, these methods comprise: (a)forming an emulsion comprising (i) a polymer selected from the groupconsisting of a poly(α-hydroxy acid), a polyhydroxy butyric acid, apolycaprolactone, a polyorthoester, a polyanhydride, and apolycyanoacrylate, (ii) an organic solvent, and (iii) water; followed by(b) removal of the organic solvent The method is carried out usingcompositions that are free of anionic surfactant, and are preferablyfree of all surfactant, including anionic, cationic, nonionic andzwitterionic surfactants.

Preferably, the emulsion is a water-in-oil-in-water emulsion that isformed by a process comprising: (a) emulsifying an organic phasecomprising polymer and organic solvent with a first aqueous phasecomprising water to form a water-in-oil emulsion; and (b) emulsifying asecond aqueous phase comprising water with the emulsion formed in step(a) to form a water-in-oil-in-water emulsion. In general, thesemicroparticle compositions are subsequently intermixed with abiologically active polypeptide-containing molecule, such as thosediscussed above, to produce a biologically active composition.

Although double-emulsion techniques like that above are preferred,single emulsion techniques can also be used to form the microparticlecompositions of the present invention.

Still other aspects of the invention are directed to methods ofproducing microparticle compositions, which methods comprise: (1)forming a microparticle in an emulsification process, whichmicroparticle comprises a polymer selected from the group consisting ofa poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone,a polyorthoester, a polyanhydride, and a polycyanoacrylate; and (2)adsorbing a biologically active polypeptide-containing molecule on thesurface of the microparticle. The method is carried out usingcompositions that are free of anionic surfactant, and are preferablyfree of all surfactant, including anionic, cationic, nonionic andzwitterionic surfactants.

An advantage of the present invention is that microparticle compositionsfor human administration, and particularly microparticle compositionsfor human administration that contain adsorbed polypeptide-containingmolecules, can be formed without resorting to the use of surfactants.The absence of surfactants is beneficial, inter alia, because theaddition of surfactants raises issues of toxicity, which issues arecircumvented by the microparticle compositions of the present invention.

These and other embodiments, aspects and advantages of the presentinvention will readily occur to those of ordinary skill in the art inview of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram of an apparatus appropriate for producingthe microparticle compositions of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, polymer chemistry,biochemistry, molecular biology, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton,Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowickand N. Kaplan, eds., Academic Press, Inc.); Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Handbook of Surface andColloidal Chemistry (Birdi, K. S., ed, CRC Press, 1997) andSeymour/Carraher's Polymer Chemistry (4th edition, Marcel Dekker Inc.,1996).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification, the singular forms “a,” “an” and “the”include plural references unless the content clearly dictates otherwise.Thus, for example, the term “microparticle” refers to one or moremicroparticles, and the like.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

Unless stated otherwise, all percentages and ratios herein are given ona weight basis.

The term “microparticle” as used herein, refers to a particle of about10 nm to about 150 μm in diameter, more preferably about 200 nm to about30 μm in diameter, and most preferably about 500 nm to about 10 μm indiameter. Preferably, the microparticle will be of a diameter thatpermits parenteral or mucosal administration without occluding needlesand capillaries. Microparticle size is readily determined by techniqueswell known in the art, such as photon correlation spectroscopy, laserdiffractometry and/or scanning electron microscopy. The term “particle”may also be used to denote a microparticle as defined herein.

Polymer microparticles for use herein are formed from materials that aresterilizable, non-toxic and biodegradable. Such materials include,without limitation, poly(α-hydroxy acid), polyhydroxybutyric acid,polycaprolactone, polyorthoester, polyanhydride, PACA, andpolycyanoacrylate. Preferably, microparticles for use with the presentinvention are polymer microparticles derived from a poly(α-hydroxyacid), in particular, from a poly(lactide) (“PLA”) or a copolymer ofD,L-lactide and glycolide or glycolic acid, such as apoly(D,L-lactide-co-glycolide) (“PLG”), or a copolymer of D,L-lactideand caprolactone. The polymer microparticles may be derived from any ofvarious polymeric starting materials which have a variety of molecularweights and, in the case of the copolymers such as PLG, a variety oflactide:glycolide ratios, the selection of which will be largely amatter of choice, depending in part on the coadministeredpolypeptide-containing molecule. These parameters are discussed morefully below.

The term “surfactant” as used herein includes detergents, dispersingagents, suspending agents, and emulsion stabilizers. Anionic surfactantsthat have been proposed in the past for use in microparticleformulations include, but are not limited to, SDS (sodium dodecylsulfate), SLS (sodium lauryl sulfate), DSS (disulfosuccinate), sulphatedfatty alcohols, and the like. Cationic surfactants that have beenproposed include, but are not limited to, cetrimide (cetyl trimethylammonium bromide, or “CTAB”), benzalkonium chloride, DDA (dimethyldioctodecyl ammonium bromide), DOTAP(dioleoyl-3-trimethylammonium-propane), and the like. Nonionicsurfactants that have been proposed include, but are not limited to,PVA, povidone (also known as polyvinylpyrrolidone or PVP), sorbitanesters, polysorbates, polyoxyethylated glycol monoethers,polyoxyethylated alkyl phenols, poloxamers, and the like.

As used herein, a composition is “free of surfactant” or there is an“absence of surfactant” within a composition where the compositioncontains only insignificant or impurity amounts of a surfactant. As usedherein an “insignificant” amount of surfactant means that thecomposition contains a weight-to-weight surfactant-to-polymer ratio ofless than 0.00001:1.

The term “macromolecule” as used herein refers to, without limitation, apharmaceutical, a polynucleotide, a polypeptide, apolypeptide-containing molecule, a hormone, an enzyme, a transcriptionor translation mediator, an intermediate in a metabolic pathway, animmunomodulator, an antigen, an adjuvant, or combinations thereof.

The term “pharmaceutical” refers to biologically active compounds suchas antibiotics, antiviral agents, growth factors, hormones, and thelike, discussed in more detail below.

The term “adjuvant” refers to any substance that assists or modifies theaction of a pharmaceutical, including but not limited to immunologicaladjuvants, which increase or diversify the immune response to anantigen.

A “polynucleotide” is a nucleic acid polymer, which typically encodes abiologically active (e.g., immunogenic or therapeutic) protein orpolypeptide. Depending on the nature of the polypeptide encoded by thepolynucleotide, a polynucleotide can include as little as 10nucleotides, e.g., where the polynucleotide encodes an antigen.Furthermore, a “polynucleotide” can include both double- andsingle-stranded sequences and refers to, but is not limited to, cDNAfrom viral, prokaryotic or eukaryotic mRNA, genomic RNA and DNAsequences from viral (e.g. RNA and DNA viruses and retroviruses) orprokaryotic DNA, and especially synthetic DNA sequences. The term alsocaptures sequences that include any of the known base analogs of DNA andRNA. The term further includes modifications, such as deletions,additions and substitutions (generally conservative in nature), to anative sequence, preferably such that the nucleic acid molecule encodesa therapeutic or antigenic protein. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the antigens.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include modifications,such as deletions, additions and substitutions (generally conservativein nature), to a native sequence, preferably such that the proteinmaintains the ability to elicit an immunological response or have atherapeutic effect on a subject to which the protein is administered.

By “antigen” is meant a molecule that contains one or more epitopescapable of stimulating a host's immune system to make a cellularantigen-specific immune response when the antigen is presented inaccordance with the present invention, or a humoral antibody response.An antigen may be capable of eliciting a cellular or humoral response byitself or when present in combination with another molecule. Normally,an epitope will include between about 3-15, generally about 5-15, aminoacids. Epitopes of a given protein can be identified using any number ofepitope mapping techniques, well known in the art. See, e.g., EpitopeMapping Protocols in Methods in Molecular Biology, Vol.66 (Glenn E.Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linearepitopes may be determined by, e.g., concurrently synthesizing largenumbers of peptides on solid supports, the peptides corresponding toportions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated hereinby reference in their entireties. Similarly, conformational epitopes arereadily identified by determining spatial conformation of amino acidssuch as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra.

The term “antigen” as used herein denotes both subunit antigens, i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature, as well as killed, attenuatedor inactivated bacteria, viruses, parasites or other microbes.Antibodies such as anti-idiotype antibodies, or fragments thereof, andsynthetic peptide mimotopes, which can mimic an antigen or antigenicdeterminant, are also captured under the definition of antigen as usedherein. Similarly, an oligonucleotide or polynucleotide which expressesa therapeutic or immunogenic protein, or antigenic determinant in vivo,such as in gene therapy and nucleic acid immunization applications, isalso included in the definition of antigen herein.

Further, for purposes of the present invention, antigens can be derivedfrom any of several known viruses, bacteria, parasites and fungi, aswell as any of the various tumor antigens. Furthermore, for purposes ofthe present invention, an “antigen” refers to a protein which includesmodifications, such as deletions, additions and substitutions (generallyconservative in nature), to the native sequence, so long as the proteinmaintains the ability to elicit an immunological response. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts that producethe antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto molecules present in the composition of interest. For purposes of thepresent invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTLs”). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote theintracellular destruction of intracellular microbes, or the lysis ofcells infected with such microbes. Another aspect of cellular immunityinvolves an antigen-specific response by helper T-cells. Helper T-cellsact to help stimulate the function, and focus the activity of,nonspecific effector cells against cells displaying peptide antigens inassociation with MHC molecules on their surface. A “cellular immuneresponse” also refers to the production of cytokines, chemokines andother such molecules produced by activated T-cells and/or other whiteblood cells, including those derived from CD4+ and CD8+ T-cells.

A composition, such as an immunogenic composition, or vaccine thatelicits a cellular immune response, may serve to sensitize a vertebratesubject by the presentation of antigen in association with MHC moleculesat the cell surface. The cell-mediated immune response is directed at,or near, cells presenting antigen at their surface. In addition,antigen-specific T-lymphocytes can be generated to allow for the futureprotection of an immunized host.

The ability of a particular antigen or composition to stimulate acell-mediated immunological response may be determined by a number ofassays, such as by lymphoproliferation (lymphocyte activation) assays,CTL cytotoxic cell assays, by assaying for T-lymphocytes specific forthe antigen in a sensitized subject, or by measurement of cytokineproduction by T cells in response to restimulation with antigen. Suchassays are well known in the art. See, e.g., Erickson et al., J.Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol (1994)24:2369-2376.

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

A composition which contains a selected antigen adsorbed to amicroparticle, displays “enhanced immunogenicity” when it possesses agreater capacity to elicit an immune response than the immune responseelicited by an equivalent amount of the antigen when delivered withoutassociation with the microparticle. Thus, a composition may display“enhanced immunogenicity” because the antigen is more stronglyimmunogenic by virtue of adsorption to the microparticle, or because alower dose of antigen is necessary to achieve an immune response in thesubject to which it is administered. Such enhanced immunogenicity can bedetermined by administering the microparticle/antigen composition, andantigen controls to animals and comparing, for example, antibody titersagainst the two using standard assays such as radioimmunoassay andELISAs, well known in the art.

The terms “effective amount,” “therapeutically effective amount” or“pharmaceutically effective amount” of a composition as provided herein,refer to a sufficient amount of the composition to treat or diagnose acondition of interest. For example, these expressions may refer to anamount sufficient to provide a desired response, such as animmunological response, and a corresponding prophylactic or therapeuticeffect, or in the case of delivery of a therapeutic protein, an amountsufficient to effect treatment of the subject, as defined below. As willbe pointed out below, the exact amount required will vary from subjectto subject, depending on the species, age, and general condition of thesubject, the severity of the condition being treated, the particularpolypeptide of interest, mode of administration, and the like. Anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

By “vertebrate subject” is meant any member of the subphylum cordata,including, without limitation, mammals such as cattle, sheep, pigs,goats, horses, and humans; domestic animals such as dogs and cats; andbirds, including domestic, wild and game birds such as cocks and hensincluding chickens, turkeys and other gallinaceous birds. The term doesnot denote a particular age. Thus, both adult and newborn animals areintended to be covered.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable. Forexample, a “pharmaceutically acceptable” material may be administered toan individual along with the microparticle formulation without causingany undesirable biological effects in the individual or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

The term “excipient” refers to substances that are commonly providedwithin finished dosage forms, and include vehicles, binders,disintegrants, fillers (diluents), lubricants, glidants (flowenhancers), compression aids, colors, sweeteners, preservatives,suspensing/dispersing agents, film formers/coatings, flavors andprinting inks.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, “treatment” (including variations thereof, for example,“treat” or “treated”) refers to any of (i) the prevention of infectionor reinfection, as in a traditional vaccine, (ii) the reduction orelimination of symptoms, and (iii) the substantial or completeelimination of the pathogen or disorder in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

As used herein, the phrase “nucleic acid” refers to DNA, RNA, orchimeras formed therefrom.

As used herein, the phrase “oligonucleotide comprising at least one CpGmotif” refers to a polynucleotide comprising at least one CpGdinucleotide. Oligonucleotides comprising at least one CpG motif cancomprise multiple CpG motifs. These oligonucleotides are also known as“CpG oligonucleotides” in the art. As used herein, the phrase “CpGmotif” refers to a dinucleotide portion of an oligonucleotide whichcomprises a cytosine nucleotide followed by a guanosine nucleotide.5-methylcytosine can also be used in place of cytosine.

According to some embodiments of the present invention, compositions andmethods are provided which treat, including prophylactically and/ortherapeutically immunize, a host animal against viral, fungal,mycoplasma, bacterial, or protozoan infections, as well as to tumors.The methods of the present invention are useful for conferringprophylactic and/or therapeutic immunity to a mammal, preferably ahuman. The methods of the present invention can also be practiced onmammals other than humans, including biomedical research applications.

B. General Methods

Surprisingly, the present inventors have found that microparticles canbe formed, and excellent adsorption of polypeptide-containing moleculesto the microparticles can be achieved, without the use of surfactants.As a result, the microparticle/polypeptide-containing-moleculecompositions of the present invention can be used as a delivery systemto deliver biologically active polypeptide-containing molecules to asubject in order to prophylactically or therapeutically treat and/ordiagnose a wide variety of diseases. While not wishing to be bound bytheory, it is believed that the polymer materials used in connectionwith the present invention (e.g., PLG) typically have negatively chargedgroups, which give the microparticles of the present invention a netnegative charge. This net negative charge leads to inter-microparticlerepulsion, stabilizing the microparticles upon their formation.Moreover, this charge also attracts positively charged regions of thepolypeptide-containing molecules, improving the adsorption of thepolypeptide-containing molecules to the microparticles.

Many exemplary embodiments within the present patent application aredirected to compositions containing microparticles with adsorbedpolypeptide-containing molecules.

The present invention can be used in connection with the delivery of awide variety of macromolecules including, but not limited to,pharmaceuticals such as antibiotics and antiviral agents, nonsteroidalantiinflammatory drugs, analgesics, vasodilators, cardiovascular drugs,psychotropics, neuroleptics, antidepressants, antiparkinson drugs, betablockers, calcium channel blockers, bradykinin inhibitors,ACE-inhibitors, vasodilators, prolactin inhibitors, steroids, hormoneantagonists, antihistamines, serotonin antagonists, heparin,chemotherapeutic agents, antineoplastics and growth factors, includingbut not limited to PDGF, EGF, KGF, IGF-1 and IGF-2, FGF, polynucleotideswhich encode therapeutic or immunogenic proteins, immunogenic proteinsand epitopes thereof for use in vaccines, hormones including peptidehormones such as insulin, proinsulin, growth hormone, GHRH, LHRH, EGF,somatostatin, SNX-111, BNP, insulinotropin, ANP, FSH, LH, PSH and hCG,gonadal steroid hormones (androgens, estrogens and progesterone),thyroid-stimulating hormone, inhibin, cholecystokinin, ACTH, CRF,dynorphins, endorphins, endothelin, fibronectin fragments, galanin,gastrin, insulinotropin, glucagon, GTP-binding protein fragments,guanylin, the leukokinins, magainin, mastoparans, dermaseptin, systemin,neuromedins, neurotensin, pancreastatin, pancreatic polypeptide,substance P, secretin, thymosin, and the like, enzymes, transcription ortranslation mediators, intermediates in metabolic pathways,immunomodulators, such as any of the various cytokines includinginterleukin-1, interleukin-2, interleukin-3, interleukin-4, andgamma-interferon, antigens, and adjuvants.

The present invention is particularly well suited for the delivery ofpolypeptide-containing molecules to a subject. In some particularlypreferred embodiments, the polypeptide-containing molecules arepolypeptide antigen molecules. One advantage of microparticles withadsorbed polypeptide antigen molecules is their demonstrated ability togenerate cell-mediated immune responses in a vertebrate subject. Thus,in addition to a conventional antibody response, the system hereindescribed can provide for, e.g., the association of the expressedantigens with class I MHC molecules such that an in vivo cellular immuneresponse to the antigen of interest can be mounted which stimulates theproduction of CTLs to allow for future recognition of the antigen.Furthermore, the methods may elicit an antigen-specific response byhelper T-cells. Accordingly, the methods of the present invention willfind use with any polypeptide-containing molecule for which cellularand/or humoral immune responses are desired, preferably antigens derivedfrom viral and bacterial pathogens that may induce antibodies, T-cellhelper epitopes and T-cell cytotoxic epitopes. Such antigens include,but are not limited to, those encoded by human and animal viruses andcan correspond to either structural or non-structural proteins.

Hence, the ability of the antigen/microparticles of the invention toelicit a cell-mediated immune response against a selected antigenprovides a powerful tool against infection by a wide variety ofpathogens. Accordingly, the antigen/microparticle compositions of thepresent invention can be incorporated into vaccine compositions.

The microparticles of the present invention are particularly useful forimmunuization against intracellular viruses which normally elicit poorimmune responses. For example, the present invention will find use forstimulating an immune response against a wide variety of polypeptidesfrom the herpes virus family, including proteins derived from herpessimplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteinsgB, gD and gH; antigens derived from varicella zoster virus (VZV),Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMV gB andgH; and antigens derived from other human herpes viruses such as HHV6and HHV7. (See, e.g. Chee et al., Cytomegaloviruses (J. K. McDougall,ed., Springer-Verlag 1990) pp. 125-169, for a review of the proteincoding content of cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988)69:1531-1574, for a discussion of the various HSV-1 encoded proteins;U.S. Pat. No. 5,171,568 for a discussion of HSV-1 and HSV-2 gB and gDproteins and the genes encoding therefor; Baer et al., Nature (1984)310:207-211, for the identification of protein coding sequences in anEBV genome; and Davison and Scott, J Gen. Virol. (1986) 67:1759-1816,for a review of VZV.)

Antigens from the hepatitis family of viruses, including hepatitis Avirus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the deltahepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus(HGV), can also be conveniently used in the techniques described herein.By way of example, the viral genomic sequence of HCV is known, as aremethods for obtaining the sequence. See, e.g., International PublicationNos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodesseveral viral proteins, including E1 (also known as E) and E2 (alsoknown as E2/NSI) and an N-terminal nucleocapsid protein (termed “core”)(see, Houghton et al., Hepatology (1991) 14:381-388, for a discussion ofHCV proteins, including E1 and E2). Each of these proteins, as well asantigenic fragments thereof, will find use in the present compositionand methods.

Similarly, the sequence for the δ-antigen from HDV is known (see, e.g.,U.S. Pat. No. 5,378,814) and this antigen can also be conveniently usedin the present composition and methods. Additionally, antigens derivedfrom HBV, such as the core antigen, the surface antigen, sAg, as well asthe presurface sequences, pre-S1 and pre-S2 (formerly called pre-S), aswell as combinations of the above, such as sAg/pre-S1, sAg/pre-S2,sAg/pre-S1/pre-S2, and pre-S1/pre-S2, will find use herein. See, e.g.,“HBV Vaccines—from the laboratory to license: a case study” in Mackett,M. and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176,for a discussion of HBV structure; and U.S. Pat. Nos. 4,722,840,5,098,704, 5,324,513, incorporated herein by reference in theirentireties; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum etal., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991)65:5457-5464.

Antigens derived from other viruses will also find use in thecompositions and methods of the present invention, such as withoutlimitation, proteins from members of the families Picomaviridae (e.g.,polioviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella virus,dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae;Bimaviridae; Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae;Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytialvirus, etc.); Orthomyxoviridae (e.g., influenza virus types A, B and C,etc.); Bunyaviridae; Arenaviridae; Retroviradae (e.g., HTLV-I; HTLV-II;HIV-I (also known as HTLV-II, LAV, ARV, hTLR, etc.)), including but notlimited to antigens from the isolates HIV_(IIIb), HIV_(SF2), HIV_(LAV),HIV_(LA1), HIV_(MN)); HIV-1_(CM235), HIV-1_(US4); HIV-2; simianimmunodeficiency virus (SIV) among others. Additionally, antigens mayalso be derived from human papillomavirus (HPV) and the tick-borneencephalitis viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed.1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe,eds. 1991), for a description of these and other viruses.

More particularly, the gp 120 or gp 140 envelope proteins from any ofthe above HIV isolates, including members of the various geneticsubtypes of HIV, are known and reported (see, e.g., Myers et al., LosAlamos Database, Los Alamos National Laboratory, Los Alamos, N. Mex.(1992); Myers et al., Human Retroviruses and Aids, 1990, Los Alamos, N.Mex.: Los Alamos National Laboratory; and Modrow et al., J Virol. (1987)61:570-578, for a comparison of the envelope sequences of a variety ofHIV isolates) and antigens derived from any of these isolates will finduse in the present methods. Furthermore, the invention is equallyapplicable to other immunogenic proteins derived from any of the variousIIV isolates, including any of the various envelope proteins such asgp160 and gp41, gag antigens such as p24gag and p55gag, as well asproteins derived from the pol and tat regions.

Influenza virus is another example of a virus for which the presentinvention will be particularly useful. Specifically, the envelopeglycoproteins HA and NA of influenza A are of particular interest forgenerating an immune response. Numerous HA subtypes of influenza A havebeen identified (Kawaoka et al., Virology (1990) 179:759-767; Webster etal., “Antigenic variation among type A influenza viruses,” p. 127-168.In: P. Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses.Springer-Verlag, New York). Thus, proteins derived from any of theseisolates can also be used in the compositions and methods describedherein.

The compositions and methods described herein will also find use withnumerous bacterial antigens, such as those derived from organisms thatcause diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis,and other pathogenic states, including, without limitation, Bordetellapertussis, Neisseria meningitides (A, B, C, Y), Neisseria gonorrhoeae,Helicobacter pylori, and Haemophilus influenza. Hemophilus influenzatype B (HIB), Helicobacter pylori, and combinations thereof. Examples ofantigens from Neisseria meningitides B are disclosed in the followingco-owned patent applications: PCT/US99/09346; PCT IB98/01665; and PCTIB99/00103. Examples of parasitic antigens include those derived fromorganisms causing malaria and Lyme disease.

Additional antigens, which are not necessarily exclusive of those listedelsewhere in this application, include the following:

-   -   A protein antigen from N. meningitidis serogroup B, such as        those in Refs. 1 to 7 below.    -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in Refs. 8, 9, 10, 11 etc.        below.    -   a saccharide antigen from N. meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in Ref. 12 below        from serogroup C (see also Ref. 13).    -   a saccharide antigen from Streptococcus pneumnoniae (e.g. Refs.        14, 15, 16).    -   an antigen from N. gonorrhoeae (e.g., Refs. 1, 2, 3).    -   an antigen from Chlamydia pneumoniae (e.g., Refs. 17, 18, 19,        20, 21, 22, 23).    -   an antigen from Chlamydia trachomatis (e.g. Ref. 24).    -   an antigen from hepatitis A virus, such as inactivated virus        (e.g., Refs. 25, 26).    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens (e.g., Refs. 26, 27).    -   an antigen from hepatitis C virus (e.g. Ref. 28).    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemaglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 (e.g., Refs. 29 & 30).    -   a diphtheria antigen, such as diphtheria toxoid (e.g., chapter 3        of Ref. 31) e.g. the CRM₁₉₇ mutant (e.g., Ref. 32).    -   a tetanus antigen, such as a tetanus toxoid (e.g., chapter 4 of        Ref. 31).    -   a protein antigen from Helicobacter pylori such as CagA (e.g.        Ref. 33), VacA (e.g. Ref. 33), NAP (e.g. Ref. 34), HopX (e.g.        Ref. 35), HopY (e.g. Ref. 35) and/or urease.    -   a saccharide antigen from Haemophilus influenzae B (e.g. Ref.        13).    -   an antigen from Porphyramonas gingivalis (e.g. Ref. 36).    -   polio antigen(s) (e.g. Refs. 37, 38) such as IPV or OPV.    -   rabies antigen(s) (e.g. Ref. 39) such as lyophilized inactivated        virus (e.g. Ref. 40, Rabavert™).    -   measles, mumps and/or rubella antigens (e.g., chapters 9, 10 and        11 of Ref. 31).    -   influenza antigen(s) (e.g. chapter 19 of Ref. 31), such as the        haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis (e.g., Ref. 41).    -   an antigen from Streptococcus agalactiae (Group B streptococcus)        (e.g. Refs. 42, 43)    -   an antigen from Streptococcus pyogenes (Group A streptococcus)        (e.g. Refs. 43, 44, 45).    -   an antigen from Staphylococcus aureus (e.g. Ref. 46).    -   compositions comprising one or more of these antigens.

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier protein in order to enhance immunogenicity (e.g.Refs. 47 to 56). Preferred carrier proteins are bacterial toxins ortoxoids, such as diphtheria or tetanus toxoids. The CRM₁₉₇ diphtheriatoxoid is particularly preferred. Other suitable carrier proteinsinclude N. meningitidis outer membrane protein (e.g. Ref. 57), syntheticpeptides (e.g. Refs. 58, 59), heat shock proteins (e.g. Ref. 60),pertussis proteins (e.g. Refs. 61, 62), protein D from H. Influenzae(e.g. Ref. 63), toxin A or B from C. difficile (e.g. Ref. 64), etc.Where a mixture comprises capsular saccharides from both serogroups Aand C, it is preferred that the ratio (w/w) of MenA saccharide:MenCsaccharide is greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher).Saccharides from different serogroups of N. meningitidis may beconjugated to the same or different carrier proteins.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or means (Ref. 30).

See: International patent application 99/24578 (Ref. 1); Internationalpatent application WO99/36544 (Ref. 2); International patent applicationWO99/57280 (Ref. 3); International patent application WO00/22430 (Ref.4); Tettelin et al., (2000) Science 287:1809-1815 (Ref. 5);International patent application WO96/29412 (Ref. 6); Pizza el al.(2000) Science 287:1816-1820 (Ref. 7); International patent applicationPCT/IB01/00166 (Ref. 8); Bjune et al. (1991) Lancet 338(8775):1093-1096(Ref. 9); Fukasawa et al. (1990) Vaccine 17:2951-2958 (Ref. 10);Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333 (Ref. 11);Costantino et al. (1992) Vaccine 10:691-698 (Ref. 12); Costantino et al.(1999) Vaccine 17:1251-1263 (Ref. 13); Watson (2000) Padiatr Infect DisJ 19:331-332 (Ref. 14); Rubin (2000) Pediatr Clin North Am 47:269-285, v(Ref. 15); Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207 (Ref. 16);International patent application filed on Jul. 3rd, 2001 claimingpriority from GB-0016363.4 (Ref. 17); Kalman et al. (1999) NatureGenetics 21 :385-389 (Ref. 18); Read et al. (2000) Nucleic Acids Res28:1397-406 (Ref. 19); Shirai et al. (2000) J. Infect. Dis. 181(Suppl3):S524-S527 (Ref. 20); International patent application WO99/27105(Ref. 21); International patent application WO00/27994 (Ref. 22);International patent application WO00/37494 (Ref. 23); Internationalpatent application WO99/28475 (Ref. 24); Bell (2000) Pediat-Infect Dis J19:1187-1188 (Ref. 25); Iwarson (1995) APMIS 103:321-326 (Ref 26);Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80 (Ref. 27); Hsu etal. (1999) Clin Liver Dis 3:901-915 (Ref. 28); Gustafssonetal. (1996) N.Engl. J. Med. 334:349-355 (Ref. 29); Rappuoli et al. (1991) TIBTECH9:232-238 (Ref. 30); Vaccines (1988) eds. Plotkin & Mortimer. ISBN0-7216-1946-0 (Ref. 31); Del Guidice et al. (1998) Molecular Aspects ofMedicine 19:1-70 (Ref. 32); International patent application WO93/18150(Ref. 33); International patent application WO99/53310 (Ref. 34);International patent application WO98/04702 (Ref. 35); Ross et al.(2001) Vaccine 19:4135-4142 (Ref. 36); Sutter et al. (2000) Pediatr ClinNorth Am 47:287-308 (Ref. 37); Zimmerman & Spann (1999) Am Fam Physicial59:113-118, 125-126 (Ref. 38); Dreesen (1997) Vaccine 15 Suppl:S2-6(Ref. 39); MMWR Morb Mortal Wkly Rep 1998 January 16;47(1):12, 19 (Ref.40); McMichael (2000) Vaccine 19 Suppl 1:S 101-107 (Ref. 41); Schuchat(1999) Lancet 353(9146):51-6 (Ref. 42); GB patent applications0026333.5, 0028727.6 & 0105640.7 (Ref. 43); Dale (1999) Infect Dis ClinNorth Am 13:22743, viii (Ref. 44); Ferretti et al. (2001) PNAS USA98:4658-4663 (Ref. 45); Kuroda et al. (2001) Lancet 357(9264):1225-1240;see also pages 1218-1219 (Ref. 46); Ramsay et al. (2001) Lancet357(9251):195-196 (Ref 47); Lindberg (1999) Vaccine 17 Suppl 2:S28-36(Ref. 48); Buttery & Moxon (2000) J R Coll Physicians London 34:163-168(Ref. 49); Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133,vii (Ref. 50); Goldblatt (1998) J. Med. Microbiol. 47:563-567 (Ref. 51);European patent 0 477 508 (Ref. 52); U.S. Pat. No. 5,306,492 (Ref. 53);International patent application WO98/42721 (Ref. 54); ConjugateVaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol.10:48-114 (Ref. 55); Hermanson (1996) Bioconjugate Techniques ISBN:0123423368 & 012342335X (Ref. 56); European patent application 0372501(Ref. 57); European patent application 0378881 (Ref 58); European patentapplication 0427347 (Ref. 59); International patent applicationWO93/17712 (Ref. 60); International patent application WO98/58668 (Ref.61); European patent application 0471177 (Ref. 62); International patentapplication WO00/56360 (Ref. 63); international patent applicationWO00/61761 (Ref. 64).

Where diphtheria antigen is included in the composition it is preferredalso to include tetanus antigen and pertussis antigens. Similarly, wherea tetanus antigen is included it is preferred also to include diphtheriaand pertussis antigens. Similarly, where a pertussis antigen is includedit is preferred also to include diphtheria and tetanus antigens.

It is readily apparent that the present invention can be used to delivera wide variety of polypeptide-containing molecules and hence to treatand/or diagnose a large number of diseases. In some embodiments, thepolypeptide-containing-molecule/microparticle compositions of thepresent invention can be used for site-specific targeted delivery. Forexample, intravenous administration of thepolypeptide-containing-molecule/microparticle compositions can be usedfor targeting the lung, liver, spleen, blood circulation, or bonemarrow.

The adsorption of polypeptide-containing molecules to the surface of theadsorbent microparticles occurs via any bonding-interaction mechanism,including, but not limited to, ionic bonding, hydrogen bonding, covalentbonding, Van der Waals bonding, and bonding throughhydrophilic/hydrophobic interactions.

Biodegradable polymers for manufacturing microparticles for use with thepresent invention are readily commercially available from, e.g.,Boehringer Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham,Ala. For example, useful polymers for forming the microparticles hereininclude homopolymers, copolymers and polymer blends derived from thefollowing: polyhydroxybutyric acid (also known as polyhydroxybutyrate);polyhydroxy valeric acid (also known as polyhydroxyvalerate);polyglycolic acid (PGA) (also known as polyglycolide): polylactic acid(PLA) (also known as polylactide); polydioxanone; polycaprolactone;polyorthoester; and polyanhydride. More preferred are poly(α-hydroxyacid), such as poly(L-lactide), poly(D,L-lactide) (both known as “PLA”herein), poly(hydoxybutyrate), copolymers of D,L-lactide and glycolide,such as poly(D,L-lactide-co-glycolide) (designated as “PLG” herein) or acopolymer of D,L-lactide and caprolactone. Particularly preferredpolymers for use herein are PLA and PLG polymers. These polymers areavailable in a variety of molecular weights, and the appropriatemolecular weight for a given use is readily determined by one of skillin the art. Thus, e.g., for PLA, a suitable molecular weight will be onthe order of about 2000 to 5000. For PLG, suitable molecular weightswill generally range from about 10,000 to about 200,000, preferablyabout 15,000 to about 150,000.

If a copolymer such as PLG is used to form the microparticles, a varietyof lactide:glycolide molar ratios will find use herein and the ratio islargely a matter of choice, depending in part on the coadministeredpolypeptide-containing molecule and the rate of degradation desired. Forexample, a 50:50 PLG polymer, containing 50% D,L-lactide and 50%glycolide, will provide a fast resorbing copolymer while 75:25 PLGdegrades more slowly, and 85:15 and 90:10, even more slowly, due to theincreased lactide component. It is readily apparent that a suitableratio of lactide:glycolide is easily determined by one of skill in theart based, for example, on the nature of the antigen and disorder inquestion. Degradation rate of the microparticles of the presentinvention can also be controlled by such factors as polymer molecularweight and polymer crystallinity. PLG copolymers with varyinglactide:glycolide ratios and molecular weights are readily availablecommercially from a number of sources including from BoehringerIngelheim, Germany and Birmingham Polymers, Inc., Birmingham, Ala. Someexemplarly PLG copolymers include: (a) RG 502, a PLG having a 50:50lactide/glycolide molar ratio and a molecular weight of 12,000 Da; (b)RG 503, a PLG having a 50:50 lactide/glycolide molar ratio and amolecular weight of 34,000 Da; (c) RG 504, a PLG having a 50:50lactide/glycolide molar ratio and a molecular weight of 48,000 Da, (d)RG 752, a PLG having a 75:25 lactide/glycolide molar ratio and amolecular weight of 22,000 Da; and (e) RG 755, a PLG having a 75:25lactide/glycolide molar ratio and a molecular weight of 68,000 Da. PLGpolymers can also be synthesized by simple polycondensation of thelactic acid component using techniques well known in the art, such asdescribed in Tabata et al., J. Biomed. Abater. Res. (1988) 22:837-858.Presently preferred PLG copolymers are those having a molarlactide/glycolide ratio ranging from 25:75 to 75:25, more preferably40:60 to 60:40, and having a molecular weight ranging from 10,000 to100,000 Daltons, more preferably from 20,000 Daltons to 70,000 Daltons.

The microparticles are prepared using any of several methods well knownin the art. For example, in some embodiments, double emulsion/solventevaporation techniques, such as those described in U.S. Pat. No.3,523,907 and Ogawa et al., Chem Pharm. Bull. (1988) 36:1095-1103, canbe used herein to make the microparticles. These techniques involve theformation of a primary emulsion consisting of droplets of polymersolution, which is subsequently mixed with a continuous aqueous phasecontaining a particle stabilizer/surfactant.

In other embodiments, microparticles can also be formed usingspray-drying and coacervation as described in, e.g., Thomasin et al., J.Controlled Release (1996) 41:131; U.S. Pat. No. 2,800,457; Masters, K.(1976) Spray Drying 2nd Ed. Wiley, New York; air-suspension coatingtechniques, such as pan coating and Wurster coating, as described byHall et al., (1980) The “Wurster Process” in Controlled ReleaseTechnologies. Methods, Theory, and Applications (A. F. Kydonieus, ed.),Vol. 2, pp. 133-154 CRC Press, Boca Raton, Fla. and Deasy, P. B., Crit.Rev. Ther. Drug Carrier Syst. (1988) S(2):99-139; and ionic gelation asdescribed by, e.g., Lim et al., Science (1980) 210:908-910.

In preferred embodiments, a modified water-in-oil-in-water (w/o/w)solvent evaporation technique can be used to form the microparticles.Techniques of this type have been described, for example, in O'Hagan etal., Vaccine (1993) 11:965-969, PCT/US99/17308 (WO 00/06123) to O'Haganet al., and Jeffery et al., Pharm. Res. (1993) 10:362. These techniques,however, are modified for use in connection with the present invention.Specifically, distinct from these techniques, the w/o/w emulsions of thepresent invention are preferably formed in the absence of surfactants(including detergents, dispersing agents, suspending agents and emulsionstabilizers).

More specifically, a particular polymer of interest such as PLG, isdissolved in an organic solvent, such as ethyl acetate, dimethylchloride (also called methylene chloride and dichloromethane),acetonitrile, acetone, chloroform, and the like. The polymer willtypically be provided in about a 1-30%, preferably about a 2-15%, morepreferably about a 3-10% and most preferably, about a 4-6% solution, inorganic solvent. The polymer solution is then combined with a firstvolume of an aqueous solution and emulsified to form an o/w emulsion.The aqueous solution can be, for example, deionized water, normalsaline, or a buffered solution such as phosphate-buffered saline (PBS)or a sodium citrate/ethylenediaminetetraacetic acid (sodiumcitrate/ETDA) buffer solution. The latter solutions can (a) provide atonicity, i.e., osmolality, that is essentially the same as normalphysiological fluids and (b) maintain a pH compatible with normalphysiological conditions. Alternatively, the tonicity and/or pHcharacteristics of the compositions of the present invention can beadjusted after microparticle formation and prior to administration.

Preferably, the volume ratio of polymer solution to aqueous solutionranges from about 5:1 to about 20:1, and is more preferably about 10:1.Emulsification is preferably conducted using any equipment appropriatefor this task, and is typically a high-shear device such as, e.g., anhomogenizer.

A volume of the o/w emulsion is then preferably combined with a largersecond volume of aqueous solution, which can also be, for example,deionized water, normal saline, or a buffered solution. The ratio of thesecond volume of aqueous solution to the volume of the o/w emulsiontypically ranges from about 2:1 to 10:1, and is more typically about4:1. The mixture is then homogenized to produce a w/o/w double emulsion.Organic solvents are then evaporated.

The formulation parameters can be manipulated to allow the preparationof small microparticles on the order of 0.2 μm (200 nm) to largermicroparticles 50 μm or even larger. See, e.g., Jeffery et al., Pharm.Res. (1993) 10:362-368; McGee et al., J. Microencap. (1996). Forexample, reduced agitation results in larger microparticles, as does anincrease in internal phase volume and an increase in polymerconcentration. Small particles are produced by increased agitation aswell as low aqueous phase volumes and low polymer concentration.

One preferred apparatus for performing the above steps is schematicallyillustrated in FIG. 1. Referring now to FIG. 1, a manufacturing tankassembly, generally designated by the numeral 102, is shown. The tankassembly 102 is designed to be a “closed system,” such that an asepticenvironment is maintained during processing. All pieces of equipment andparts are preferably selected to be clean-in-place and autoclavable. Allfilters 104 a-d are preferably fluoropolymer filters such asSuper-Cheminert™ all-fluoropolymer filters from Pall Corporation.Initially, an aqueous solution, such as a deionized water 106 and anorganic polymer solution, such as a solution of PLG in methylenechloride 108, are filtered and fed into tank 110 where they arecontinuously mixed with mixer 112. The mixture is then fed through anin-line homogenizer 114 (e.g., a high speed, high shear autoclavablein-line homogenizer such as the Kinematica MT 5000), forming an o/wemulsion. The emulsion is cooled, for example by a water-cooledcondenser 116, after emerging from the in-line homogenizer 114,whereupon it is returned to the tank 110. After the contents areemulsified to the desired extent, additional aqueous solution, such asdeionized water 106, is added to the tank 110, whereupon a w/o/wemulsion is formed by again feeding the contents through the in-linemixer 114. The resulting w/o/w emulsion is purged with nitrogen viadistributor 119 to remove the organic solvent. The nitrogen-ladensolvent vapor is filtered and cooled in a condenser 120, capturing thesolvent in container 122. Where the emulsion is somewhat unstable, itmay be desirable to remove the solvent concurrently with in-line mixing.

Particle size can be determined by, e.g., laser light scattering, usingfor example, a spectrometer incorporating a helium-neon laser.Generally, particle size is determined at room temperature and involvesmultiple analyses of the sample in question (e.g., 5-10 times) to yieldan average value for the particle diameter. Particle size is alsoreadily determined using scanning electron microscopy (SEM).

Following preparation, microparticles can be stored as is or lyophilizedfor future use. In order to adsorb polypeptide-containing molecules tothe microparticles, the microparticle preparation can be simply mixedwith the polypeptide-containing molecule of interest and the resultingformulation can again be lyophilized prior to use.

Typically, polypeptide-containing molecules are added to themicroparticles to yield microparticles with adsorbedpolypeptide-containing molecules having a polypeptide-containingmolecule to microparticle weight-to-weight ratio of from about 0.0001:1to 0.25:1, more typically 0.001:1 to 0.1:1, even more typically 0.05:1to 0.01:1. The polypeptide-containing-molecule content of themicroparticles can be determined using standard techniques.

In addition to microparticles with adsorbed polypeptide-containingmolecules, the compositions of the present invention can also include avariety of other macromolecules (including additionalpolypeptide-containing molecules, pharmaceuticals, polynucleotides,hormones, enzymes, transcription or translation mediators, metabolicpathway intermediates, immunomodulators, antigens, adjuvants orcombinations thereof.) For example, the microparticles of the presentinvention may have additional macromolecules entrapped or encapsulatedwithin them, adsorbed on their surfaces, or included in solution or insuspension. Particularly preferred additional macromolecules areadjuvants.

Once the microparticles with adsorbed polypeptide-containing moleculesare produced, they are formulated into pharmaceutical compositions,including vaccines, to treat and/or diagnose a wide variety ofdisorders, as described above. The compositions will generally includeone or more pharmaceutically acceptable excipients. For example,vehicles such as water, saline, glycerol, polyethylene glycol,hyaluronic acid, ethanol, etc. may be used. Other excipients, such aswetting or emulsifying agents, biological buffering substances, and thelike, may be present in such vehicles. A biological buffer can bevirtually any substance which is pharmacologically acceptable and whichprovides the formulation with the desired pH, i.e., a pH in thephysiological range. Examples of buffer solutions include phosphatebuffered saline (PBS), Tris buffered saline, Hank's buffered saline, andthe like. Other excipients known in the art can also be introduced intothe final dosage form, including binders, disintegrants, fillers(diluents), lubricants, glidants (flow enhancers), compression aids,colors, sweeteners, preservatives, suspensing/dispersing agents, filmformers/coatings, flavors and printing inks.

Adjuvants may be used to enhance the effectiveness of the pharmaceuticalcompositions. The adjuvants may be administered concurrently with themicroparticles of the present invention, e.g., in the same compositionor in separate compositions. Alternatively, the adjuvant may beadministered prior or subsequent to the microparticle compositions ofthe present invention. In some embodiments, the adjuvant, such as animmunological adjuvant, may be encapsulated in the microparticle.Adjuvants, just as any macromolecule, may be encapsulated within themicroparticles using any of the several methods known in the art. See,e.g., U.S. Pat. No. 3,523,907; Ogawa et al., Chem. Pharm. Bull. (1988)36:1095-1103; O'Hagan et al., Vaccine (1993) 11:965-969 and Jefferey etal., Pharm. Res. (1993) 10:362. Alternatively, some adjuvants,particularly polypeptide-containing adjuvants, may be adsorbed on themicroparticle as described above.

Immunological adjuvants include, but are not limited to: (1) aluminumsalts (alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) other oil-in water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (International Publication No. WO90/14837; Chapter 10 in Vaccinedesign: the subunit an adjuvant approach, Eds. Powell & Newman, PlenumPress 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE (see below), althoughnot required) formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b)SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP (see below) either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion, and(c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cellwall components from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™) (for a further discussion of suitable submicronoil-in-water emulsions for use herein, see commonly owned, patentapplication Ser. No. 09/015,736, filed on Jan. 29, 1998); (3) saponinadjuvants, such as Quil A, or QS21 (e.g., Stimulon™ (CambridgeBioscience, Worcester, Mass.)) may be used or particles generatedtherefrom such as ISCOMs (immunostimulating complexes), which ICOMS maybe devoid of additional detergent e.g., WO00/07621; (4) Complete FreundsAdjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines,such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12(WO99/44636), etc.), interferons (e.g. gamma interferon), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.;(6) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g.GB-2220221, EP-A-0689454, optionally in the substantial absence of alumwhen used with pneumococcal saccharides e.g. WO00/56358; (7)combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions, e.g., EP-A-0835318, EP-A-0735898, EP-A-076123 1; (8)oligonucleotides comprising CpG motifs (Roman et al., Nat. Med., 1997,3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis etal., J Immunol. 1988, 160, 870-876; Chu et al., J. Exp. Med., 1997, 186,1623-1631; Lipford et al., Eur. J. Immunol. 1997, 27, 2340-2344;Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature,1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883:Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J.Immunol., 1996, 156, 45704575; Halpern et al., Cell. Immunol., 1996,167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873;Stacey et al., J. Immunol, 1996, 157,2116-2122; Messina et al., J.Immunol, 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157,4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J.Immunol., 1998, 160, 47554761; and Yi et al., J. Immunol., 1998, 160,5898-5906; International patent applications WO96/02555, WO98/16247,WO98/18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581) i.e.containing at least one CG dinucleotide, with 5 methylcytosineoptionally being used in place of cytosine; (9) a polyoxyethylene etheror a polyoxyethylene ester e.g. WO99/52549; (10) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (WO01/21207)or a polyoxyethylene alkyl ether or ester surfactant in combination withat least one additional non-ionic surfactant such as an octoxynol(WO01/21152); (11) a saponin and an immunostimulatory oligonucleotide(e.g., a CpG oligonucleotide) (WO00/62800); (12) an immunostimulant anda particle of metal salt e.g. WO00/23105; (13) a saponin and anoil-in-water emulsion e.g. WO99/11241; (14) a saponin (e.g.QS21)+3dMPL+IL-12 (optionally+a sterol) e.g. WO98/57659; (15) detoxifiedmutants of a bacterial ADP-ribosylating toxin such as a cholera toxin(CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT),particularly LT-K63 (where lysine is substituted for the wild-type aminoacid at position 63) LT-R72 (where arginine is substituted for thewild-type amino acid at position 72), CT-S 109 (where serine issubstituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. WO93/13202 and WO92/19265); (16)aminoalkyl glucosaminide 4-phosphates (AGP's), see, e.g., Johnson, D. A.et al.; Bioorg. Med. Chem. Lett., 1999 Aug. 2; 9(15):2273-8, (17)imidazoquinolines such as imiquimod (R-837) and resiquimod (R-848), see,e.g., Vasilakos, J. P. et al.; Cell. Immunol. 2000 Aug. 25;204(l):64-74, (18) lipopolysaccharide mimetics, including non-saccharidephospholipids (e.g., simplified lipid A analogs lacking a disaccharide)described in Hawkins, L. D. et al; J. Pharmacol. Exp. Ther., 2002February; 300(2):655-61, and (19) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-nomuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

For additional examples of adjuvants, see Vaccine Design:, The Subunitand the Adjuvant Approach, Powell, M. F. and Newman, M. J, eds., PlenumPress, 1995)

The compositions will comprise a “therapeutically effective amount” ofthe polypeptide-containing molecule (as well as any other macromolecule)of interest. That is, a sufficient amount of the polypeptide-containingmolecule will be included to treat or diagnose a condition of interest.The exact amount necessary will vary, for example, depending on thesubject being treated; the age and general condition of the subject tobe treated; the severity of the condition being treated; in the case ofan immunological response, the capacity of the subject's immune systemto synthesize antibodies; the degree of protection desired and theparticular polypeptide-containing molecule selected and its mode ofadministration, among other factors. An appropriate effective amount canbe readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will typically fall in a relativelybroad range that can be determined through routine trials. For example,where the macromolecule is a polypeptide antigen, an effective dose willtypically range from about 1 μg to about 100 mg, preferably from about 5μg to about 1 mg, more preferably about 5 μg to about 100 μg and mostpreferably about 5 μg to about 50 μg of the antigen delivered per dose.

Once formulated, the compositions of the invention can be administeredparenterally, e.g., by injection. The compositions can be injectedeither subcutaneously, intraperitoneally, intravenously orintramuscularly. Other modes of administration include nasal, mucosal,rectal, vaginal, oral and pulmonary administration, suppositories, andtransdermal or transcutaneous applications.

Dosage treatment may be a along a single dose schedule or a multipledose schedule. A multiple dose schedule is one in which a primary courseof administration may be with 1-10 separate doses, followed by otherdoses given at subsequent time intervals, chosen to maintain and/orreinforce the therapeutic response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the subject and be dependent on the judgment of thepractitioner.

Furthermore, if prophylactic treatment is desired, for example, invaccines, compositions of the present invention are generallyadministered prior to primary infection with the pathogen of interest.If therapeutic treatment is desired, e.g., the reduction of symptoms orrecurrences, the compositions of the present invention are generallyadministered subsequent to primary infection.

C. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Preparation of PLG Particles with No Surfactant

2.5 ml PBS is homogenized with 10 ml 6% RG503 (a PLG Polymer having a50:50 lactide/glycolide molar ratio and a molecular weight of 34,000Daltons, available from Boehringer Ingelheim) in dimethyl chloride witha small probe of the IKA homogenizer (Germany) at 23,000 rpm for 2minutes. This primary o/w emulsion is added to 50 ml of deionized water,followed by homogenization with a 10 mm probe of the Omni benchtophomogenizer (LabTek Inc, US) at 15,000 rpm for 30 minutes in an icebath. The container is sealed using Teflon tape with the homogenizerinserted into the liquid to prevent solvent evaporation duringhomogenization. The Teflon tape is removed, and this secondary w/o/wemulsion is left stirring overnight to allow for solvent evaporation.The next day the particle size is measured using a Malvern Master Sizer.The size range is typically 0.5-1 micron.

EXAMPLE 2 Preparation of PLG Particles with 0.05% and 0.5% wt:wt DSS toPLG

2.5 ml PBS is homogenized with 10 ml 6% RG503 (a PLG Polymer having a50:50 lactide/glycolide molar ratio and a molecular weight of 34,000Daltons, available from Boehringer Ingelheim) in dimethyl chloride witha small probe of the IKA homogenizer (Germany) at 23,000 rpm for 2minutes. This primary o/w emulsion is added to 50ml of deionized water,containing either 6 ug/ml or 60 ug/ml DSS for 0.05% and 0.5%respectively. This is followed by homogenization with a 10 mm probe ofthe Omni benchtop homogenizer (LabTek Inc, US) at 15,000 rpm for 30minutes in an ice bath. The container is sealed using Teflon tape withthe homogenizer inserted into the liquid to prevent solvent evaporationduring homogenization. The Teflon tape is removed, and this secondaryw/o/w emulsion is left stirring overnight to allow for solventevaporation. The next day the particle size is measured using a MalvernMaster Sizer. The size range is typically 0.5-1 micron.

EXAMPLE 3 Preparation of PLG Particles (Conventional Formulation)

2.5 ml PBS is homogenized with 10 ml 6% RG503 (a PLG Polymer having a50:50 lactide/glycolide molar ratio and a molecular weight of 34,000Daltons, available from Boehringer Ingelheim) in dimethyl chloride witha small probe of the IKA homogenizer (Germany) at 23,000 rpm for 2minutes. This primary o/w emulsion is added to 50 ml of deionized water,containing 1% wt:vol DSS. This is followed by homogenization with a 10mm probe of the Omni benchtop homogenizer (LabTek Inc, US) at 10,000 rpmfor 3 minutes at room temperature. This secondary w/o/w emulsion is leftstirring overnight to allow for solvent evaporation. The next day theparticle size is measured using a Malvern Master Sizer. The size rangeis typically 0.5-1 micron.

EXAMPLE 4 Protein Adsorption to PLG Formulations

PLG particles made with 0%, 0.05% and 0.5% wt:wt DSS (from Examples 1and 2 above) were adsorbed to meningitis B 287 protein, (Chiron proteinpurification group, Siena, Italy. Vol. 287 Science, 1816 (2000)) asfollows: Direct binding to PLG particles with 0%, 0.05% and 0.5% DSS:

-   1—The suspension volume for each formulation in Examples 1 and 2,    was measured and PLG content was estimated by dividing the starting    weight of PLG by the total volume.-   2—For each formulation, a specific volume containing 200 mg of PLG    was placed in a 30 ml centrifuge tube and 2 mg of 287 protein were    added.-   3—The buffer was adjusted to 10 mM Citrate by adding 1 ml of 100 mM    Citrate pH4.75 and the total volume was brought up to 10 ml with DI    water.-   4—The tube was left rocking on a lab rocker at 4° C. overnight.-   5—The next day, a 2 ml aliquot was withdrawn for analysis and the    remaining suspension was aliquot into vials, each containing 12    doses of either lug or 10 ug of protein on PLG per dose.-   6—216 ul of 25% wt:vol solution of Mannitol in water were added to    each vial prior to lyophilization.

EXAMPLE 5 Protein Adsorption to PLG/DSS Formulation made by theConventional Method

-   1—The suspension in Example 3 was washed with 250 ml water by    centrifugation twice.-   2—The pellet is resuspended in 15 ml DI water and sonicated in a    water bath sonicator for 2 minutes.-   3-5 ml of the suspension (containing 200 mg PLG ) was placed in a 30    ml centrifuge tube and 2 mg 287 protein was added.-   4—The buffer was adjusted by adding 1 ml 10×PBS and the volume    brought up to 10 ml with DI water.-   5—The suspension was allowed to rock on a lab rocker overnight at 4°    C.-   6—The next day a 1 ml aliquot was withdrawn for analysis and the    remaining suspension was transferred to a 12-14M wt. Cut off    dialysis tubing and dialyzed against 4 changes of 4 L DI water each.-   7—The total volume was measured and a 1 ml aliquot was withdrawn for    analysis. The remaining suspension was aliquoted into vials, each    containing either 1 ug or 10 ug protein on PLG.-   8—216 ul of a 25% solution of Mannitol in DI water was added to each    vial before lyophilization.

EXAMPLE 6 Characterization

-   1—1 ml of each suspension (from Example 4, step 5) was dialyzed in a    12-14 M wt. Cut off dialysis tubing against 2 changes of 4 L water    each, overnight, and lyophilized.-   2—The remaining 1 ml of each suspension (from Example 4, step 5) and    the aliquot from Example 5 step 6, were each washed with 30 ml water    by centrifugation and lyophilized.-   3—1 ml aliquot from Example 5, step 7 was lyophilized.-   4—5 mg of each lyophilized formulation from steps 1, 2 and 3 above,    were hydrolyzed with 1 ml each 0.2 NNaOH/5% SDS, and protein content    measured by Micro BCA assay from Pierce, USA.-   5—10 mg each of unwashed particles (from steps 1 and 3 above) were    resuspended in 1 ml PBS and left rocking at 37° C. for one hour and    burst release was determined by measuring protein in the supernatant    by Micro BCA assay from Pierce, USA.

Unwashed Washed Post-adsorption Post-adsorption % load % 1 hour Sizemicrons Formulation % load wt:wt wt:wt release D50-90 0% DSS/287 0.63 165 0.82-1.2  0.05% DSS/287 0.85 1 56 0.9-1.5 0.5% DSS/287 0.61 1 293.8-9.3 Conventional 0.87 1 40  9-28 formulation/287

EXAMPLE 7 In Vivo Data

Groups of 10 CD 1 mice each, were immunized intramuscularly with 100 ulcontaining either 10 ug or 1 ug of protein adsorbed onto PLG particlesfrom Example 4, step 5 and Example 5, step 7 at 0, 3 and 5 weekintervals and sera was collected at weeks 5 and 7.

Enzyme-linked immunosorbent assay designed to measure MenB-specificantibody was performed on mice sera at week 5 and 7. Purified 287protein was coated onto Nunc Maxisorp U bottom plates (Nalgene NuncInternational) at 1 ug/ml. Sera were tested at 1:100 and 1:400 dilutionsfollowed by serial three fold dilutions. Horseradishperoxidase-conjugated goat anti mouse IgG (CALTAG diluted 1:40,000) wasused as a second antibody. After the one-hour incubation at 37° C.,plates were washed to remove unbound antibody. TMB (Kirkegaard and PerryLaboratories, KPL) substrate was used to develop the plates and thecolor reaction was blocked after 15 minutes by addition of 2N HCl. Thetiters reported, geometric mean titer (GMT) along with standard error(STE), are the reciprocal of the serum dilutions that gave an opticaldensity at 450 nm of 0.5 ELISA absorbency units.

2 weeks post 2nd 2 weeks post 3rd Formulation Dose GMT STE GMT STE   0%DSS/287  1 ug 407 331 1,159 2,389 10 ug 4,324 1,771 7,138 2,223 0.05%DSS/287  1 ug 889 390 3,314 473 10 ug 7,586 3,494 10,274 3,016  0.5%DSS/287  1 ug 1,380 2,369 2,316 1,253 10 ug 1,963 1,573 4,551 1,175Conventional  1 ug 334 510 1,288 586 formulations 10 ug 1,779 1,3404,020 1,457

Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of theinvention.

1. A microparticle composition comprising: (a) microparticles comprisinga polymer selected from the group consisting of a poly(α-hydroxy acid),a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, apolyanhydride, and a polycyanoacrylate; and (b) a polypeptide-containingmolecule adsorbed to the microparticles, wherein the microparticlecomposition is formed in the absence of surfactant and wherein themicroparticles do not contain entrapped or encapsulatedpolypeptide-containing molecules.
 2. The microparticle composition ofclaim 1, wherein the polymer comprises a poly(α-hydroxy acid).
 3. Themicroparticle composition of claim 2, wherein the poly(α-hydroxy acid)is selected from the group consisting of poly(L-lactide),poly(D,L-lactide) and poly(D,L-lactide-co-glycolide).
 4. Themicroparticle composition of claim 3, wherein the polymer comprisespoly(D,L-lactide-co-glycolide).
 5. The microparticle composition ofclaim 4, wherein the poly(D,L-lactide-co-glycolide) has alactide/glycolide molar ratio ranging from 25:75 to 75:25 and amolecular weight ranging from 10,000 to 100,000 Daltons.
 6. Themicroparticle composition of claim 4, wherein thepoly(D,L-lactide-co-glycolide) has a lactide/glycolide molar ratioranging from 40:60 to 60:40 and a molecular weight ranging from 20,000Daltons to 70,000 Daltons.
 7. The microparticle composition of any ofclaims 1-6, wherein the polypeptide-containing molecule is an antigen.8. The microparticle composition of claim 7, wherein the antigen isselected from HIV antigens, meningitis B antigens, streptococcusantigens, hepatitis B virus antigens, hepatitis C virus antigens,Haemophilis influenza type B antigens, pertussis antigens, diphtheriaantigens, tetanus antigens, Helicobacter pylori antigens and Influenza Ahemagglutinin antigens.
 9. The microparticle composition of claim 7,wherein the antigen is selected from the group consisting of HIV gp41antigen, HIV gp120 antigen, HIV gp140 antigen, HIV p24gag antigen, HIVp55gag antigen, meningitis B recombinant protein 287 antigen, and groupB streptococcus antigen.
 10. The microparticle composition of claim 1,further comprising a pharmaceutically acceptable excipient.
 11. Themicroparticle composition of claim 10, further comprising an additionalbiologically active macromolecule selected from the group consisting ofa polynucleotide, a polynucleoside, a pharmaceutical, a hormone, anenzyme, a transcription or translation mediator, an intermediate in ametabolic pathway, an immunomodulator, and an adjuvant.
 12. Themicroparticle composition of claim 11, wherein the additionalbiologically active macromolecule is an adjuvant.
 13. The microparticlecomposition of claim 12, wherein the adjuvant is a member selected fromthe group consisting of CpG oligonucleotides, double-stranded RNAadjuvants, aminoalkyl glucosaminide 4-phosphate adjuvants,imidazoquinoline adjuvants, lipopolysaccharide mimetic adjuvants,saponin adjuvants, E. coli heat-labile toxin adjuvants,monophosphorylipid A adjuvants and aluminum salts.
 14. The microparticlecomposition of claim 12, wherein the adjuvant is aluminum phosphate. 15.A method of delivering a therapeutically effective amount of apolypeptide-containing molecule to a vertebrate subject, the methodcomprising the step of administering to the vertebrate subject themicroparticle composition of any of claims 10-14.
 16. The microparticlecomposition of claim 7, wherein the antigen comprises a polysaccharideconjugated to a polypeptide.
 17. A method of producing a microparticlecomposition, the method comprising: (a) forming microparticles by asurfactant-free emulsification process, the microparticles comprising apolymer selected from the group consisting of a poly(α-hydroxy acid), apolyhydroxy butyric acid, a polycaprolactone, a polyorthoester, apolyanhydride, and a polycyanoacrylate; and (b) adsorbing apolypeptide-containing molecule on the surface of the microparticles toform the microparticle composition.
 18. The method of claim 17, whereinthe emulsification process comprises: (a) forming an emulsion comprisingan organic solvent, water and the polymer; and (b) removing the organicsolvent from the emulsion to form microparticles.
 19. The method ofclaim 18, wherein the emulsion is a water-in-oil-in-water emulsion thatis formed by a process comprising: (a) emulsifying an organic phasecomprising the polymer and the organic solvent with a first aqueousphase comprising water to form a water-in-oil emulsion; and (b)emulsifying a second aqueous phase comprising water with the emulsionformed in step (a) to form a water-in-oil-in-water emulsion.
 20. Themethod of claim 19, wherein the emulsifying steps are conducted in ahigh-shear homogenizer.
 21. The method of any of claims 17-20, whereinthe polymer is a poly(α-hydroxy acid).
 22. The method of any of claims17-20, wherein the polymer is a poly(D,L-lactide-co-glycolide).
 23. Themethod of claim 22, wherein the poly(D,L-lactide-co-glycolide) has alactide/glycolide molar ratio ranging from 25:75 to 75:25 and amolecular weight ranging from 10,000 to 100,000 Daltons.
 24. The methodof claim 22, wherein the polymer is a poly(D,L-lactide-co-glycolide)having a lactide/glycolide molar ratio ranging from 40:60 to 60:40 and amolecular weight ranging from 20,000 Daltons to 70,000 Daltons.
 25. Themethod of any of claims 17-20, wherein the polypeptide-containingmolecule is an antigen.
 26. The method of claim 25, wherein the antigenis selected from HIV antigens, meningitis B antigens, streptococcusantigens, hepatitis B virus antigens, hepatitis C virus antigens,Haemophilus influenza type B antigens, pertussis antigens, diphtheriaantigens, tetanus antigens, Helicobacter pylori antigens and Influenza Ahemagglutinin antigens.
 27. The method of claim 25, wherein the antigenis selected from the group consisting of HIV gp41 antigen, HIV gp120antigen, HIV gp140 antigen, HIV p24gag antigen, HIV p55gag antigen,meningitis B recombinant protein 287 antigen, and group B streptococcusantigen.
 28. A microparticle composition formed by a process comprising:(a) forming microparticles by a surfactant-free emulsification process,the microparticles comprising a polymer selected from the groupconsisting of a poly(α-hydroxy acid), a polyhydroxy butyric acid, apolycaprolactone, a polyorthoester, a polyanhydride, and apolycyanoacrylate; and (b) adsorbing a polypeptide-containing moleculeon the surface of the microparticles to form the microparticlecomposition.
 29. The method of any of claims 17-20, wherein aweight-to-weight ratio of the adsorbed polypeptide-containing moleculeto the polymer ranges between 0.001:1 and 0.1:1.
 30. The microparticlecomposition of any of claims 1-6, and 10-13, wherein a weight-to-weightratio of the adsorbed polypeptide-containing molecule to the polymerranges between 0.01:1 and 0.05:1.
 31. The microparticle composition ofclaim 28, wherein the polymer comprises a poly(α-hydroxy acid).
 32. Themicroparticle composition of claim 31, wherein the poly(α-hydroxy acid)is selected from the group consisting of poly(L-lactide),poly(D,L-lactide) and poly(D,L-lactide-co-glycolide).
 33. Themicroparticle composition of claim 28, wherein thepolypeptide-containing molecule is an antigen.
 34. The microparticlecomposition of claim 33, wherein the antigen is selected from HIVantigens, meningitis B antigens, streptococcus antigens, hepatitis Bvirus antigens, hepatitis C virus antigens, Haemophilus influenza type Bantigens, pertussis antigens, diphtheria antigens, tetanus antigens,Helicobacter pylori antigens and Influenza A hemagglutinin antigens. 35.The microparticle composition of claim 33, wherein the antigen isselected from the group consisting of HIV gp41 antigen, HIV gp120antigen, HIV gp140 antigen, HIV p24gag antigen, HIV p55gag antigen,meningitis B recombinant protein 287 antigen, and group B streptococcusantigen.
 36. The microparticle composition of claim 28, furthercomprising an additional biologically active macromolecule selected fromthe group consisting of a polynucleotide, a polynucleoside, apharmaceutical, a hormone, an enzyme, a transcription or translationmediator, an intermediate in a metabolic pathway, an immunomodulator,and an adjuvant.
 37. The microparticle composition of claim 36, whereinthe additional biologically active macromolecule is an adjuvant.
 38. Themicroparticle composition of claim 37, wherein the adjuvant is a memberselected from the group consisting of CpG oligonucleotides,double-stranded RNA adjuvants, aminoalkyl glucosaminide 4-phosphateadjuvants, imidazoquinoline adjuvants, lipopolysaccharide mimeticadjuvants, saponin adjuvants, E. coil heat-labile toxin adjuvants,monophosphorylipid A adjuvants and aluminum salts.
 39. The microparticlecomposition of claim 28, wherein the emulsification process comprises:(a) forming an emulsion comprising an organic solvent, water and thepolymer; and (b) removing the organic solvent from the emulsion to formmicroparticles.
 40. The microparticle composition of claim 39, whereinthe emulsion is a water-in-oil-in-water emulsion that is formed by aprocess comprising: (a) emulsifying an organic phase comprising thepolymer and the organic solvent with a first aqueous phase comprisingwater to form a water-in-oil emulsion; and (b) emulsifying a secondaqueous phase comprising water with the emulsion formed in step (a) toform a water-in-oil-in-water emulsion.
 41. The microparticle compositionof claim 40, wherein the emulsifying steps are conducted in ahomogenizer.